NASBA testing for rapid detection of West Nile and St. Louis encephalitis viruses
West Nile and St. Louis encephalitis viruses are arthropod-borne viruses of the Flaviviridae family. West Nile virus historically has circulated primarily in Africa, Asia, southern Europe, and Australia and has been responsible for several significant epidemics. In 1999 and 2000, West Nile virus was responsible for epidemics and epizootics in the northeastern United States in which there were human fatalities and extensive avian mortality. St. Louis encephalitis virus is endemic throughout the United States and has also been isolated in several South American countries. St. Louis encephalitis virus has during the past 70 years been responsible for numerous epidemics throughout the United States, the largest of these occurring in 1975, with approximately 2,000 cases reported. Timely implementation of public education and mosquito control programs are important in reducing the risk to humans. Surveillance programs for these viruses typically involve field-collected mosquito testing and, in the case of West Nile virus, isolating the virus in the cell culture of dead birds. In the clinical laboratory, human West Nile virus and St. Louis encephalitis virus infections can be inferred by immunoglobulin M (IgM) capture in IgG enzyme-linked immunosorbent assays. Confirmation of the type of infecting virus, however, is possible only by detecting a four-fold or greater rise in viral-specific neutralizing antibody titers in cerebrospinal fluid or serum by performing the plaque-reduction neutralization assay with several flaviviruses. Viral isolation in cell culture from CSF or serum generally has been unsuccessful. Several investigators have reported on TaqMan assays for detecting West Nile virus in human CSF specimens for which cell culture assays were negative, suggesting that nucleic acid-based assays hold greater promise for detecting these viruses in human specimens. The authors developed and applied the technique of nucleic acid sequence-based amplification (NASBA) assays for detecting West Nile and St. Louis encephalitis viruses. They developed two unique detection formats for the NASBA assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (NASBA-ECL assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (NASBA-beacon assay). The sensitivities and specificities of these two assays were compared to those of the newly described standard reverse transcription PCR (RT-PCR) and TaqMan assays for St. Louis encephalitis virus and to the previously published TaqMan assay for West Nile virus. The NASBA assays demonstrated exceptional sensitivities and specificities compared to those of virus isolation, the TaqMan assays, and standard RT-PCR, with the NASBA beacon assay yielding results in less than one hour. The authors concluded that these assays should be useful in the diagnostic laboratory, complementing existing diagnostic technologies for flavivirus surveillance in the United States.

Lanciotti RS, Kerst AJ. Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses. J Clin Microbiol. 2001; 12:4506-4513.

Reprints: Robert S. Lanciotti, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, CDC, Rampart Rd., Ft. Collins, CO 80521; rsl2@cdc.gov

EXCEL throat culture proficiency testing
The original intent of proficiency testing under CLIA '88 was to assess laboratory performance and, potentially, to improve the quality of testing services as labs received feedback on their performance and gained experience through such testing. An example of such a testing program is the EXCEL PT program operated by the College of American Pathologists. The author examined whether the hypothesized improvement due to proficiency testing participation and feedback was occurring. The author chose a problem from the EXCEL throat culture module that involved differentiating group A from non-group A streptococci, specifically the recognition of group C streptococci. This was selected because of its clinical significance in ruling out preventable sequelae. The author examined the ability of participants in the EXCEL program to differentiate group A streptococci from group C streptococci over a six-year period from 1996 through 2001. He specifically looked for changes in participant performance levels. Feedback on performance relative to peers and an educational discussion analyzing performance and suggesting best practices was submitted to participants after each testing cycle. In spite of this consistent feedback, however, there was no significant change in participant performance throughout the study period. The author concluded that use of proficiency testing results in laboratory improvement programs is suboptimal.

Novak RW. Do proficiency testing participants learn from their mistakes? Experience from the EXCEL throat culture module. Arch Pathol Lab Med. 2002;126:147-149.

Reprints: Dr. Robert Novak, Dept. of Pathology, Children's Hospital, 1 Perkins Square, Akron, OH 44308; rnovak@chmca.org

C-reactive protein as a predictor for diabetes mellitus
C-reactive protein is a sensitive marker for systemic inflammation. It has been reported to be elevated in patients with cardiovascular disease and other inflammatory conditions. Since inflammation may be an important antecedent for the development of type 2 diabetes mellitus, the authors prospectively examined the association of C-reactive protein (CRP) and diabetes mellitus in middle-aged men. They studied 2,052 initially nondiabetic men, aged 45 to 74 years, who were part of a large cohort of men. Diabetes was diagnosed through self-reported questionnaires. Serum CRP determinations (nonfasting) were measured using an immunoradiometric assay. Samples were stored frozen (-80�C) until analysis. Lipid profiles were also measured. Incidence rates for diabetes mellitus were age standardized. During an average followup of 7.2 years (range, 0.1 to 13.7 years), 101 new cases of diabetes were detected. Subjects with hypertension, history of myocardial infarction, or angina had significantly higher geometric mean CRP concentrations compared with those who did not have these disorders. Smoking was also strongly associated with elevated serum CRP levels (almost a two-fold increased level in smokers compared with those who never smoked). There was a significant positive association between elevated CRP levels and diabetes. This association, however, became nonsignificant when adjusted for body mass index, smoking status, and systolic blood pressure. The authors speculated that these factors played a causal role in the induction of an inflammatory state and the subsequent development of type 2 diabetes mellitus in men. Inflammation could be one mechanism by which known risk factors for diabetes mellitus, such as obesity, smoking, and hypertension, promote the development of diabetes mellitus.

Thorand B, L�wel H, Schneider A, et al. C-reactive protein as a predictor for incident diabetes mellitus among middle-aged men. Arch Intern Med. 2003;163:93-99.

Correspondence: Dr. Wolfgang Koenig, Dept. of Internal Medicine II-Cardiology, University of Ulm Medical Center, Robert Koch Str 8, D-89081 Ulm, Germany; wolfgang.koenig@medizin.uni-ulm.de

Evaluating robotic specimen preparation workstations
Automated specimen-processing units have, in recent years, been designed for the preanalytical section of the small to medium-sized laboratory, and this has affected laboratory operations. Relatively few studies of laboratory automation in the literature have provided information that focuses specifically on the preanalytic portion of specimen processing. The authors evaluated the Genesis FE500 automated preanalytical processing unit at two academic medical centers. The unit processes blood specimens through automated specimen sorting, centrifugation, decapping, labeling, aliquoting, and placement of the processed specimen in the analytical rack. The authors quantified the output of the FE500 by processing more than 3,000 bar-coded specimens according to a protocol designed to test all the features of the unit. The mean system output performance varied between 93 and 502 total tubes/h. This depended on the size of the batch, aliquot number requested, and percentage of tubes that required centrifugation. Throughput increased as batch size expanded from 40 or 100 samples (mean, 211 total tubes processed/h) to a batch size of 200 and 300 tubes (mean, 474 total tubes processed/h). The device processed tubes ranging in size from 13 ∞ 65 mm to 16 ∞ 100 mm. At one site, the FE500 was operated by a single person, whereas three people had been required to perform the same tasks manually. The specimen-processing error rate determined at one of the institutions declined significantly with use of the equipment. The authors concluded that the Genesis FE500 reduces the labor associated with specimen processing and decreases laboratory errors.

Holman JW, Mifflin TE, Felder RA, et al. Evaluation of an automated preanalytical robotic workstation at two academic health centers. Clin Chem. 2002;48:540-548.

Reprints: Dr. Laurence M. Demers, Penn State University, Departments of Pathology and Medicine, Milton S. Hershey Medical Center, P.O. Box 850, Hershey, PA 17033; lmd4@psu.edu

HIV from platelet donation
When HIV antibody screening was the only HIV test performed on blood donations, the residual risk of HIV transmission by transfusion was estimated to be approximately one in 493,000. In spite of universal screening of blood donations, it was still possible to transmit HIV via transfusion because the HIV antibody test detects only antibody formed against the virus by the infected person, not the virus itself. The period between HIV exposure and the ability to detect HIV antibody, known as the window period, consists of two phases. In the first phase, the HIV replication is occurring in lymph nodes and the liver but not in the blood. This phase is known as the eclipse phase since circulating virus cannot be demonstrated. In the second phase, known as the viremic phase, HIV is circulating but HIV antibody is not detectable. Studies have estimated the median length of the entire window period to be approximately 40 days. The viremic phase of the HIV window is approximately 22 days. Nucleic acid testing (NAT) for HIV RNA is now being performed under investigational new drug applications with the FDA. The additional effect NAT will have on reducing or better understanding the window period is not known. It has been estimated that it will reduce the window period by only an additional five days. The authors reported on the case of a 35-year-old frequent platelet donor who tested HIV p24 antigen positive and antibody negative before implementation of NAT. The subject made two platelet donations immediately before testing positive for HIV. The donor's HIV seroconversion was then monitored, and stored samples were tested retrospectively for HIV RNA. Platelet recipients were tested for HIV infection. The day-four sample tested positive for HIV RNA by pooled and individual sample NAT. The day-11 sample tested negative for HIV RNA by both NAT tests. The two recipients of the day-four platelets tested HIV RNA and p24 antigen positive. The recipient of the day-11 platelets could not be tested because he had died. HIV NAT would have prevented transmission of HIV had it been available at the time of the donor’s HIV seroconversion.

Kopko PM, Fernando LP, Bonney EN, et al. HIV transmissions from a window-period platelet donation. Am J Clin Pathol. 2001;116:562-566.

Reprints: Dr. Patricia M. Kopko, Sacramento Medical Foundation Blood Centers, 1625 Stockton Blvd., Sacramento, CA 95186-7089

Performing procedures on the newly deceased
The newly deceased may be used in teaching lifesaving procedures, such as endotracheal intubation, placement of central venous catheters, liver biopsy, and bone marrow biopsy. Such procedures also may be performed on the newly deceased in the context of an autopsy. The authors explored the ethical issues surrounding informed consent in this setting and provided recommendations. Central to the ethical debate over use of the newly deceased for training purposes is the deceased person’s claim to autonomy, respecting the wishes of the family, and the importance of properly training physicians in potentially lifesaving procedures. The concern that obtaining informed consent would decrease the number of training opportunities has not been supported in the medical literature. The majority of studies have shown that families who are appropriately and sensitively asked generally give consent. Likewise, the idea of “presumed” consent raises serious ethical concerns. Studies have also shown that medical trainees are more comfortable performing procedures on the newly deceased once they know that informed consent has been obtained. Trust in the medical profession is also enhanced when informed consent is sought. The authors concluded that physicians should work to develop institutional policies that address the practice of performing procedures on the newly deceased; informed consent should be obtained from the family before performing such procedures; such procedures should be performed in the context of structured training rather than as random opportunities; and training should be performed under close supervision and in a respectful manner and environment.

The Council on Ethical and Judicial Affairs of the American Medical Association. Performing procedures on the newly deceased. Aca Med. 2002;77:1212-1216.

Reprints: Karine Morin, LLM, Council on Ethical and Judicial Affairs, Ethics Standards Group, American Medical Association, 515 N. State St., Chicago, IL 60610; karine_morin@ama-assn.org

Monoclonal gammopathy in patients with primary cytomegalovirus infection
Monoclonal gammopathy results from a clonal proliferation of differentiated plasma cells producing homogenous immunoglobulin or its light chain. The product, M component, or paraprotein, appears in the serum or as Bence Jones proteinuria, or both. The observance of IgM-l M component in an otherwise healthy individual with primary cytomegalovirus (CMV) infection prompted this study. The M component in this person disappeared within six months. For their study, the authors analyzed three groups of patients. Group one comprised the index case and 24 other immunocompetent patients aged 17 to 62 years who had acute or recent primary CMV infection. Group two comprised 24 immunocompetent patients aged 15 to 49 years who had acute or recent primary Epstein-Barr virus (EBV) infection. Group three comprised one bone marrow and seven solid-organ transplant recipients aged 18 to 60 years who had acute or recent primary CMV infection. Serological studies were performed to diagnose primary CMV or EBV infection. M component was found by immunofixation electrophoresis in 10 (40 percent) of 25 of the immunocompetent patients (group one) with primary CMV infection and in five of eight (62.5 percent) of the transplant recipients (group three) with primary CMV infection. None of the patients in group two (EBV infection) had M component. Of the 15 patients with M component, nine had IgG-class M component, five had IgM, and one had IgA. Twelve of those 15 patients had l light-chain-type M component. Half of the 15 patients showed no abnormal peak in serum electrophoresis, and the M component was detectable only by immunofixation. In those patients with followup, M component remained detectable for up to six weeks. The authors concluded that monoclonal gammopathy is common among immunocompromised and immunocompetent patients with primary CMV infection.

B�hler S, Laitinen K, Holth�fer H, et al. High rate of monoclonal gammopathy among immunocompetent subjects with primary cytomegalovirus infection. Clin Infect Dis. 2002;35:1430-1433.

Reprints: Dr. Suvi B�hler, Helsinki University Central Hospital Laboratory Diagnostics, Dept. of Virology, P.O. Box 400, Haartmaninkatu 3, FIN-00029 HUCH, Helsinki, Finland; suvi.buhler@hus.fi